Ab7659

CD4-VioGreen (130-106-655, Miltenyi Biotec), Anti-CCR10-PE (clone: REA326, 130-104-822, Miltenyi Biotec), CD183 (CXCR3)-PE-Vio770 (clone: REA326, 130-104-822, Miltenyi Biotec), CD194 (CCR4)-APC (clone: REA279, 130-103-813, Miltenyi Biotec), CD196 (CCR6)- PE-Vivo-615 (clone: REA190, 130-107-142, Miltenyi Biotec), Propidium iodide (130-093-233, Miltenyi Biotec), NESTIN rabbit-anti-mouse (ab7659, Abcam), SCA1 rat-anti-mouse (ab25195, Abcam), PDGFRα goat-anti-mouse (AF1062, R&D Systems), Alexa Flour 405 donkey-anti-Rabbit (ab175651, Abcam), Alexa Flour 488 donkey-anti-goat (A-11055, Thermo Fisher Scientific), Alexa Flour 594 donkey-anti-rat (A-21209, Thermo Fisher Scientific), PerCP-CyTM 5.5 CD45.2 conjugated mouse-anti-mouse (552950, BD Biosciences), ICAM1 rabbit-anti-human (ab53013, Abcam), VCAM1 rabbit-anti-human (ab134047, Abcam), IL-1β rabbit-anti-human (ab2105, Abcam), TNF-α rabbit-anti-human (ab9635, Abcam), actin rabbit-anti-human (A2066, Sigma-Aldrich), rabbit anti goat (81–1620, Invitrogen), goat anti rabbit (65–6120,Invitrogen), CD3 rabbit-anti mouse (AD452, Dako), and B220 rat-anti-mouse (MAB1217, R&D Systems).

For the IHC, the primary antibodies used for immunochemistry were as follows: VEGF (Bioss, bs-1665R) and Nestin (Abcam, ab7659). Secondary antibodies were all purchased from Boster in China. In brief, the slides were deparaffinized and rehydrated by gradient elution using xylene and ethanol, followed by incubation in 3% H₂O₂ to suppress endogenous peroxidase activity. For antigen retrieval, the slides were incubated in hyaluronidase (Sigma-Aldrich, St Louis, MO, USA) for 1 h at 37 °C. After the sealing of 5% BSA, the specimens were incubated with primary antibody diluted 1:100 at 4 °C overnight. Then sections were then rinsed in PBS and incubated with biotinylated secondary antibody for 20 min at room temperature. SABC kit purchased from Boster (Wuhan, China) was used for the staining process. 3,3′-Diaminobenzidine (DAB) was used as a color developing agent, and then slides were counterstained with hematoxylin. Finally, slides were mounted with Permount TM Mounting Medium and observed by the stereomicroscope (Carl Zeiss, Stemi 508, Germany) and the microscope (Nikon Eclipse 80i, Japan). Pictures were modified by PhotoshopCS6 (Adobe, CA, USA). Negative controls were incubated with normal anti-rabbit or anti-mouse IgG instead of the primary antibodies.

The human NPCs were fixed with 4% paraformaldehyde (PFA, Synth, Diadema, SP, Brazil, 01P1005.01) for 1 h at 4 °C, permeabilized and blocked with 5% BSA solution (LGC, Sao Paulo, SP, Brazil, 9048-46-8) and 0.5% TritonX-100 (LGC, Sao Paulo, SP, Brazil, 13-1315-05) for 30′ at room temperature. Primary antibodies were diluted in blocking solution and labeled overnight at 4 °C. The following day, secondary antibodies diluted in blocking solution were used for 2 h in the dark at room temperature. Finally, DAPI (Invitrogen, Itapevi, SP, Brazil, D1306) was used to label the cell nucleus at a 1:5000 dilution in 1× PBS for 10 min in the dark at room temperature. Visualization was performed using a ZOE Biorad^® (Hercules, CA, USA) microscope. Primary antibodies: anti-ZIKV envelope protein (rabbit, GeneTex, Irvine, CA, USA, GTX133314, 1:200); anti-MAP2 (chicken, Abcam, Cambrigde, UK, ab5392, 1:200); anti-SOX2 (goat, Abcam, Cambrigde, UK, ab97959, 1:200); anti-musashi-1 (goat, Abcam, Cambrigde, UK, ab52865, 1:200); anti-nestin (mouse, Abcam, Cambrigde, UK, ab7659, 1:200). Secondary antibodies: anti-chicken 488 (Invitrogen, Waltham, MA, USA, A-11039, 1:500); anti-rabbit 555 (Abcam, Cambrigde, UK, ab150078, 1:500); anti-goat 488 (Invitrogen, Waltham, MA, USA, A32184, 1:500); anti-goat 555 (Invitrogen, Waltham, MA, USA, A21432, 1:500).